Identification of GDF9 Promoter SNPs Affecting Lambing Rate in Australian-Hu Sheep

Background: Litter size represents a key economic trait in Australian-Hu sheep (Australian White sheep ♂ × Hu sheep ♀) and determines breeding profitability. Growth differentiation factor 9 (GDF9) is recognized as a principal candidate gene influencing litter size in ovis aries. Previous research on Australian-Hu sheep has focused on associations between polymorphisms in the coding region of GDF9 and litter size, with limited attention to the promoter region. To address this gap, this study examined the GDF9 promoter region polymorphism in Australian-Hu sheep, analyzed its association with multiple lambing and evaluated its impact on promoter activity, aiming to provide a theoretical foundation for selecting molecular markers for multiple lambing breeding. Methods: To investigate single nucleotide polymorphism (SNP) loci effects in GDF9 promoter region on lambing rate of Australian-Hu crossbred sheep, 193 pregnant ewes were selected. Genomic DNA (gDNA) was extracted from blood, the GDF9 promoter region was amplified by PCR and SNP sites were identified using Sanger sequencing. Association analyses between SNPs and lambing rate were conducted and a dual-luciferase reporter assay evaluated the SNPs’ effects on GDF9 transcriptional activity. Result: In the GDF9 promoter region of Australian-Hu Sheep, six linked SNP sites were identified: g.42117805C greater than T, g.42117824T greater than C, g.42117899G greater than A, g.42118070A greater than G, g.42118197T greater than G and g.42118272C greater than T. Five loci (g.42118070A greater than G, g.42118272C greater than T, g.42117805C greater than T, g.42117824T greater than C and g.42117899G greater than A) showed significant association with lambing rate (P less than 0.05), with heterozygous genotypes yielding higher lambing rate. Dual-luciferase reporter assays indicated that the (-517 to +3) region functions as the core promoter. Mutations at g.42118070A greater than G, g.42118197T greater than G and g.42118272C greater than T within this region reduced GDF9 promoter transcriptional activity. These findings provide a basis for identifying molecular markers to screen for multiple lambing traits in Australian-Hu sheep.